Translation is a central process in biology, and endpoint of gene expression. A large body of biochemical data has provided a mechanistic view of protein synthesis, which has recently been augmented by high and medium resolution structures of the ribosome and its complexes. However, dynamic information about the conformational and compositional rearrangement of the ribosome during translation is not available. Here, we build on the work of the prior funding period that established single-molecule fluorescence approaches to probe translation. In specific aim 1, we will use single-molecule fluorescence to monitor the rates and dynamics of assembly of the translational initiation complex at the start codon. The timing of factor and tRNA binding will be measured, and the fidelity of start site selection determined;effects of factor and mRNA mutations will be determined. In specific aim 2, we will broaden our prior work on tRNA selection to understand the contribution of tRNA flexibility, and the inhibition of tRNA selection by antibiotics. We will focus a variety of single-molecule fluorescence and FRET approaches on the mechanism of ribosomal translocation. We will attempt to measure the movements of tRNA, ribosomal RNA and mRNA that accompany translocation. In specific aim 3, to approach more closely true biology, we will study translational mechanism in the complex context of a cell-free translation extract;initiation and elongation rates will be measured, and effects of reaction conditions and changes in mRNA sequence monitored. Finally, specific aim 4 focuses on receding events in translation: -1 frameshifting, +1 frameshifting and ribosomal hopping. We will use single-molecule methods (in collaboration with J. Atkins, Univ of Utah) to study the role of mRNA sequence and structure on tRNA and ribosomal dynamics near and at receding sites. The results of this work will bridge the mechanistic and structural data on ribosome function, by providing a dynamic view of ribosome structure in action.